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To test the role of neuronal Dnmt1 in obesity development, we fed male and female ND1KO and fl/fl mice a 60% high fat diet (HFD; Research Diets D12492; New Brunswick, NJ) starting at 6 weeks of age. ND1KO male mice weighed significantly less after two weeks on the HFD (Fig. 6A), and continued to weigh less throughout the duration of the experiment (the maximal weight difference occurred at 11 weeks on the HFD).

Figure 6. Body weight, length and composition of HFD-fed fl/fl and ND1KO mice.

Weekly body weight (A, B), body composition (C, D) and body length (E, F) of male and female HFD-fed fl/fl and ND1KO mice. * p< .05, **p< .01, ***p< .001.

Unlike the chow-fed ND1KO mice, HFD-fed ND1KO mice had reduced body length (Fig. 6E). Male mice also had significantly reduced percentage of body fat, with a tendency for an increase in lean mass (Fig. 6C). HFD-fed female ND1KO mice had significant differences in body weight Figure 7. Fat pad and liver weights of HFD-fed fl/fl and ND1KO mice.

Epididymal/perigonadal, subcutaneous, retroperitoneal white adipose tissues, brown adipose tissue and liver weights (G, H); and fat and liver weights normalized to body weight of male and female HFD-fed fl/fl and ND1KO mice. *p< .05, **p< .01, ***p< .001.

Figure 8. Body weight distribution of HFD-fed fl/fl and ND1KO mice.

throughout the duration of the HFD as indicated by Repeated-Measures ANOVA (Fig. 6B); however, final body weight at the end of the study did not significantly differ between the Figure 9. Glucose and insulin tolerance tests (GTT/ITT) for HFD-fed fl/fl and ND1KO mice.

Blood glucose concentrations over two hours following an i.p. glucose infusion, and mean area under the curve blood glucose concentrations during the test, in male (A, C) and female (B, D) HFD-fed fl/fl and ND1KO mice. Blood glucose concentrations over two hours following an i.p. insulin infusion, and mean area under the curve of the blood glucose concentrations during the test, in male (E, G) and female (F, H) HFD-fed fl/fl and ND1KO mice. *p<.05, **p<.01, ***p< .001.

genotypes. Female ND1KO mice showed no significant differences in body composition or body length compared to fl/fl female mice (Figs. 6D, F).

HFD-fed ND1KO male mice had significantly smaller epididymal, subcutaneous and retroperitoneal fat pads, even when normalized to body weight (Fig. 7A, C). Brown adipose tissue was significantly smaller, but not when normalized to body weight. Female ND1KO mice had significantly smaller perigonadal, subcutaneous and brown adipose tissue fat pads and these differences remained significant even after body weight normalization (Fig. 7B, D). Both male and female ND1KO mice had smaller liver weights (Fig. 7A-B), although after adjusting for body weight, male ND1KO mice only had a tendency for reduced liver weight (Fig. 7A). The distribution and variability of body weight of HFD-fed males and females at 31-34 weeks of age is illustrated in Fig. 8A-B.

We tested whether neuronal Dnmt1 deletion altered glucose and insulin dynamics in the HFD-fed mice, since diet-induced obesity causes glucose and insulin intolerance. In the HFD-fed male mice, there was no change in the ability to clear a glucose infusion from the blood between the two genotypes (Fig. 9A, C). HFD-fed female ND1KO mice showed a very mild

improvement in overall glucose tolerance as seen in the area under the curve (Fig. 9B, D). In response to an insulin injection, however, male ND1KO mice had a lower area-under-the-curve (AUC) of blood glucose (Fig. 9E, G), and female ND1KO mice had a similar effect (Fig. 9F, H). Female ND1KO mice, however, showed a significant main effect of genotype throughout the duration of the insulin tolerance test, and follow-up tests at various time points revealed significant reductions in blood glucose at the 30- and 60-minute time points (Fig. 9F).

The following results pertain only to male ND1KO and fl/fl mice, since the largest phenotype was observed in only male ND1KO mice fed an HFD. We quantified serum

concentrations of insulin, leptin, non-esterified (free) fatty acids, and triglycerides of the fl/fl and ND1KO male mice (Table 1). Neuronal Dnmt1 deficiency did not alter serum leptin or insulin concentrations in chow-fed mice, although there was a trend for ND1KO mice to have lower concentrations of both hormones. HFD-fed ND1KO mice had significantly lower serum leptin concentrations compared to HFD-fed fl/fl controls, as might be expected from reduced adiposity. Leptin is secreted in direct proportion to the amount of fat stores in the body (Jequier, 2002). Serum insulin was no different in HFD-fed ND1KO or fl/fl control mice, however. Neuronal Dnmt1 deficiency did not affect serum triglycerides or non-esterified fatty acids in either chow- fed or HFD-fed mice.

Consistent with their reduced obesity, HFD-fed ND1KO male mice consumed less food over a 7-day period (Fig. 10A). We placed the male mice in metabolic cages and found that ND1KO mice had higher energy expenditure during both light and dark periods than the fl/fl mice (Fig. 10B). In addition, ND1KO mice had higher physical activity level (Fig. 10C), and a decreased respiratory exchange ratio (RER) (Fig. 10D) during one light-dark cycle of the 7-day

Table 1. Serum hormone and lipid measurements.

Serum insulin, leptin, triglycerides and non-esterified fatty acids in fed-state of chow- and HFD-fed male fl/fl and ND1KO mice. ** p< .01.

metabolic cage experiment, which indicates a preferential usage of fat acids as an energy source in ND1KO mice.